FAQs

Categories

GENERAL

Does Pacific Immunology® guarantee antibody titers?

Yes, Pacific Immunology® guarantees at least a 1:50,000 antibody titer against peptide sequences that we synthesize/conjugate. Titers are confirmed via ELISA tests that we run.

Does Pacific Immunology® offer any catalog antisera?

No, Pacific Immunology® focuses exclusively on the production of custom antisera for its clients.

Do I retain rights to the antisera that I generate?

Yes, you will receive all serum / peptides / columns used for generation of your antibody and retain rights to these materials.

Does Pacific Immunology® maintain the confidentiality of my project?

Yes, Pacific Immunology® places great importance in ensuring that our clients’ information remains confidential and will never release details of any potential, current or past projects to any outside parties.

Does Pacific Immunology® generate Monoclonal antibodies?

No, Pacific Immunology® has chosen to maintain its 40-year focus in generating custom polyclonal antibodies. Also, our Monospecific antibody production program generates epitope-specific polyclonal antibodies at a fraction of the cost of monoclonal antibody development.

What is the advantage of Pacific Immunology®'s AdjuLite™ Freund's Adjuvants?

Pacific Immunology®'s ultra pure Freund's Adjuvants are comprised of premium components that not only enhance the immune response, but significantly reduce the stress of the immunization on the animal. For example, only a single subcutaneous injection is needed per immunization (multiple site injections are avoided) and abscesses and irritation are eliminated for the animal. For additional information, please visit our AdjuLite™ Freund's Adjuvant page.

Why are your prices so competitive?

As one of the world’s largest and oldest providers of custom antibodies, we enjoy economies of scale that allow us to generate high quality antibodies at very competitive prices.

Does Pacific Immunology® offer volume discounts?

What is Sodium Azide and should I add it to the antiserum?

Sodium Azide is a preservative that prevents bacteria from growing in the serum (bacteria can produce proteases that denature any proteins in the serum, including antibodies). We typically recommend adding azide unless the serum is going to be used directly in cell culture applications. Azide can be removed by using a dialysis membrane with a molecular weight cutoff between 12,000 and 14,000.

How will the serum be shipped?

Serum is typically shipped after each bleed is taken. The serum will be frozen, spun down and shipped via FedEx overnight on ice packs.

What concentration of antibody can be expected?

In general, there will be approximately 5-10mg of IgG in each ml of serum. Of this, there will be approximately 100ug of peptide-specific antibody. So, a typical affinity purification of 25ml of serum will yield approximately 2-3mg of specific antibody.

Will the titer of antibody increase with additional immunizations?

For rabbit projects, the titers remain relatively level after the 1st production bleed and additional immunizations are used to maintain antibody titers rather than to increase them. Specificity does increase between the 1st and 3rd bleeds as part of the affinity maturation process and so antibodies from the third and later bleeds represent the most specific antibodies (which is why the affinity purification is run on the third bleed).

Are accelerated protocols available?

No, accelerated protocols are largely a marketing gimmick that overlooks the natural timeline needed for the animal’s immune system to develop high affinity antibodies.

What kind of rabbits / chickens does Pacific Immunology® use?

New Zealand White Rabbits and Rhode Island Red Chickens

Will I receive any leftover peptide at the end of the project?

Yes, you will receive all leftover peptide at the end of your project, helping ensure the confidentiality of your research. Typically we synthesize 40-50mg of peptide, so you should receive 30-45mg of peptide at the end.

ANTIGEN PREPARATION

How much protein do I need to send?

We recommend sending 2-3 mg of the antigen for a rabbit or chicken project and 5-7 mg for a goat project. We recommend .5 mg/ml as a minimum protein concentration and welcome higher concentrations.

How should I send my protein to Pacific Immunology®?

Please ship the antigen in a Styrofoam box with ice packs (dry ice isn’t necessary) via FedEx, UPS or DHL Overnight to the address listed on our Order Form. To avoid additional shipping charges by these companies, please insert the Styrofoam box into a cardboard box or shipping pak prior to shipping. Please also include a printed copy of the Order Form with the shipment.

Does the protein need to be soluble?

No, insoluble proteins can be used for immunizations.

Can I generate antisera against gel strips containing my protein?

Yes, immunizing with gel bands works well for generating polyclonal antisera against a purified protein. If providing a gel strip, please stain with coomassie blue, de-stain, rinse and cut out the strip containing the target protein. Please avoid freezing or homogenizing the gel strip so that we can aliquot appropriate quantities of the protein for each immunization. With gel bands in particular, it is important to maintain a higher concentration of the protein as outlined above.

Should I generate antibodies against a full-length protein or a peptide?

Generating antibodies against a full-length protein will provide a pool of antibodies against multiple epitopes from the protein, thereby maximizing the probability of recognizing the endogenous protein in the target assay. However, this larger pool of antibodies does increase the possibility that some of those antibodies will cross-react with other proteins in the assay. Immunizing with a peptide sequence, by contrast, allows the serum to be affinity purified against the peptide sequence, thereby permitting isolation of antibodies that are highly specific to the target protein. The only potential downside to this approach is that there is a risk that the chosen sequence won’t correspond to an exposed region of the native protein.

Does Pacific Immunology® offer antigen design assistance?

Yes, our peptide chemists will analyze your protein sequence and recommend the most immunogenic sequences based on such factors as the hydrophobicity / hydrophilicity and folding characteristics of the protein. We have consistently obtained very good results with these recommendations and provide this service at no additional charge.

Why is conjugation of the peptide to a carrier protein necessary?

The molecular weight of most peptides is too small to generate an immune response in the animal. Conjugation to a carrier protein such as KLH will not only increase the size of the antigen, but increase its immunogenicity as well.

What role does the adjuvant play and what is the difference between CFA and IFA?

An adjuvant is a substance that, when combined with the antigen, serves to enhance the immune response against the antigen. Freund's adjuvants are the preferred adjuvant of choice for use in antibody production, and Pacific Immunology® uses its own premium line of AdjuLite™ adjuvants for all immunizations. AdjuLite™ Complete Freund's Adjuvant is used only for the first immunization and contains mycobacteria, which stimulates the host's immune system. AdjuLite™ Incomplete Freund's Adjuvant is used for all subsequent immunizations.

ANTIBODY USAGE

How should I store the serum / antisera?

For short-term storage of the working antibody or of serum that has recently arrived, 2-3 weeks at 4°C is recommended. For long-term storage, we recommend storing the serum vials at -20°C, which is sufficient for several years. Storage at -80°C is fine also, but not necessary except for very long periods of time. The purified antibody should be aliquoted into working quantities and stored according to the guidelines above. The biggest threat to antibody stability is repeated freeze/thaw cycles. To avoid this, we recommend storing ready-to-use aliquots of the antibody and thawing aliquots only as they are needed. We also recommend the use of manual defrost freezers rather than frostless freezers. For more information, please visit our Antibody Storage page.

Why am I seeing multiple or unexpected bands on a Western with my affinity purified antibody?

It isn’t uncommon to see multiple bands even when using affinity-purified antibody. This isn’t indicative of a problem with the antibody’s specificity. Rather, this typically occurs for one of the following reasons: The anti-peptide antibody recognizes a homologous protein in the sample that shares one or more epitopes with the peptide sequence. The native protein is a different molecular weight than previously predicted. The antibody is recognizing either cleaved fragments of the native protein at lower molecular weights or aggregated dimers/trimers of the native protein at higher molecular weights.

Why am I not seeing any bands on my western when assaying with the purified antibody?

In cases where no bands are seen (or an antibody doesn’t work in a particular assay), these are the three most common explanations: The peptide sequence corresponds to a non-exposed region of the native protein The protein’s conformation in the peptide region differs enough that the antibody has trouble recognizing the native protein. The target protein isn’t present in the sample.

How do I interpret the ELISA results?

The ELISA measures the peptide-specific immune response. Dilutions of pre-immune serum and the first production bleed are incubated in peptide-coated microwells to determine the point where the OD reading of the production bleed is 0.1 above the background (pre-immune serum). A titer of 1:50,000 to 1:100,000 indicates a good immune response.

PURIFICATION

What is the difference between an IgG purification and an affinity purification?

An IgG purification isolates all IgG in the serum, regardless of the specificity. This is advantageous for applications where removing serum proteins will help reduce background noise, but affinity purification against the immunizing protein isn’t possible. The affinity purification, by contrast, is advantageous for isolating only those antibodies that recognize a specific epitope. With a peptide, this can yield antibodies that have the same specificity as monoclonal antibodies.

Is the affinity column included so that I can run additional purifications?

Yes, the affinity column will be shipped with the affinity-purified antibodies.

How many times can the affinity column be used?

Although the capacity does decrease slightly with each use, the column can often be used for at least 10 additional purifications. We recommend storing the column at 4o C (avoid freezing or drying) and with PBS azide (added by default). In addition, it can help to filter the serum through a membrane (.45) before running it through the column (to help prevent the build up of particulates in the column). The column should also be washed between uses to remove any protein.